Stem cells and decellularized tissue matrix from cord tissue

ABSTRACT

Methods and products obtained from the method for isolating and culturing mixed populations of stem cells, making decellularized tissue matrix, making decellularized tissue matrix infused with said mixed populations of stem cells, and methods of stem cell therapy are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.13/890,134, filed May 8, 2013, which claims priority to U.S. App. Ser.No. 61/644,423, filed May 8, 2012, each of which is expresslyincorporated by reference in its entirety for all purposes herein.

FEDERALLY SPONSORED RESEARCH STATEMENT

Not applicable.

BACKGROUND OF THE INVENTION

Stem cells are unspecialized cells that have two defining properties:the ability to differentiate into other cells and the ability toself-regenerate. The ability to differentiate is the potential todevelop into other cell types. A totipotent stem cell (e.g. fertilizedegg) can develop into all cell types including the embryonic membranes.A pluripotent stem cell can develop into cells from all three germinallayers (e.g., cells from the inner cell mass). Other cells can beoligopotent, bipotent or unipotent depending on their ability to developinto few, two or one other cell type(s). Self-regeneration is theability of stem cells to divide and produce more stem cells. Duringearly development, stem cell division is believed to be symmetrical i.e.each cell divides to give rise to daughter cells, each with the samepotential. Later in development, stem cells are believed to divideasymmetrically with one of the daughter cells produced being a stem celland the other a more differentiated cell (Sharpless N. E. and De PinhoR. A., “How stem cells age and why this makes us grow old”, Nature Rev.MCB, 2007)

Stem cells are further classified according to their differentiationpotential, roughly as follows:

Differentiation Number of Example of stem Cell types resulting Potentialcell types cell from differentiation Totipotential All Zygote(fertilized All cell types egg), blastomere Pluripotential All exceptCultured human Cells from all three cells of the ES cells germ layersembryonic membranes Multipotential Many Hematopoietic skeletal muscle,cardiac cells muscle, liver cells, all blood cells Oligopotential FewMyeloid precursor 5 types of blood cells (Monocytes, macrophages,eosinophils, neutrophils, erythrocytes) Quadripotential 4 MesenchymalCartilage cells, fat progenitor cell cells, stromal cells, bone-formingcells Tripotential 3 Glial-restricted 2 types of astrocytes, precursoroligodendrocytes Bipotential 2 Bipotential B cells, macrophagesprecursor from murine fetal liver Unipotential 1 Mast cell precursorMast cells Nullipotential None Terminally No cell divisiondifferentiated cell e.g. Red blood cell

As development proceeds, there is a loss of potential and a gain ofspecialization, a process called determination. For example, the cellsof the germ layers are more specialized than the fertilized egg or theblastomere. The germ layer stem cells give rise to progenitor cells(also known as progenitors or precursor cells). For example, a cell inthe endoderm gives rise to a primitive gut cell (progenitor), which canfurther divide to produce a liver cell (a terminally differentiatedcell).

While there is consensus in the literature that a progenitor is apartially specialized type of stem cell, there are differences in howprogenitor cell division is described. For instance, according to onesource, when a stem cell divides at least one of the daughter cells itproduces is also a stem cell; when a progenitor cell undergoes celldivision it produces two specialized cells. A different source, however,explains that a progenitor cell undergoes asymmetrical cell division,while a stem cell undergoes symmetrical cell division.

The different kinds of human stem cells identified to date include:embryonic stem cells derived from embryos artificially produced inin-vitro fertility clinics, fetal stem cells derived from abortedfetuses, umbilical cord blood and placental blood stem cells, umbilicalcord and placental tissue stem cells, bone marrow blood stem cells,peripheral blood stem cells, bone marrow mesenchymal stem cells, adultfat or adipose tissue-derived stem cells, cardiac muscle stem cells,skin epidermis stem cells, endothelial progenitor cells, brain andspinal cord derived neural stem cells, dental pulp stem cells andolfactory epithelium stem cells. In addition, human embryonic-like stemcells can be synthetically manufactured by inducing any adult terminallydifferentiated cell like a cheek skin fibroblast or even an adult stemcell into an embryonic-like stem cell.

Stem cells of the umbilical cord tissue are defined as mesenchymal stemcells or stromal stem cells with set characteristics. However, not allumbilical cord tissue cells have been tested for stem cell activity.Cord tissue mesenchymal stem cells have an ability to adhere tolaboratory flask or dish surfaces and their morphology isfibroblast-like. They are believed to express specific surface markerslike CD105, CD133, CD166, CD44, CD54, CD90, HLA-ABC, CD146, CD73, STRO-1and are capable of differentiating into chondrocytes, cardiomyocytes,adipocytes and osteocytes. Cord tissue mesenchymal stem cells areexpected to engraft better than bone marrow mesenchymal stem cells orblood stem cells because unlike older tissue or bone marrow derivedmesenchymal stem cells, they do not express a mature set of majorhistocompatibility antigens.

However, there are also inconsistencies in how stem cells are describedand/or identified, making the field a challenging one to understand, andthe literature is replete with inconsistencies. Mendicino M. et. al.,(2014) “MSC-Based Product Characterization for Clinical Trials: An FDAPerspective”, Cell Stem Cell 14, February 6.

The ability of stem cells to self-renew and give rise to subsequentgenerations with variable degrees of differentiation capacity offerssignificant potential to replace diseased and damaged areas in the body,with minimal risk of rejection and side effects. Many medicalresearchers believe that stem cell treatments have the potential tochange the face of human disease and human tissue degeneration. Althoughunidentified back then, stem cells were used in tribal medicinethousands of years ago when tribe medical leaders looked for “young”blood to treat sick tribe members that would be regarded today asconsanguineous people.

A number of stem cell therapies already exist, but most are atexperimental stages, with the notable exception of bone marrow and cordblood transplantations. Whether experimental or not, stem cell therapiesare also costly. Medical researchers anticipate that adult and embryonicstem cells will soon be able to treat cancer, Type 1 diabetes mellitus,Parkinson's disease, Huntington's disease, Celiac Disease, cardiacfailure, muscle damage and neurological disorders, and many others.Nevertheless, more research is necessary to further understand stem cellbehavior before and after processing and upon transplantation as well asthe mechanisms of stem cell interaction with the diseased/injuredmicroenvironment. Keating, A. (2012) “Mesenchymal Stromal Cells: NewDirections”, Cell Stem Cell 10, 709-716; Akkermann R., Beyer F., Miry P.(2017) “Heterogeneous populations of neural stem cells contribute tomyelin repair”, Neural Regen Res. April; 12(4):509-517.

It is also important to note that it is not yet fully understood whatfactors define the environment of a cell or what factors define thenormal biology of different cells during development, adulthood andaging. However, it is clear that ontogeny and age-related phenomenaexert an effect on cells and environment thereof. Stem cells ofdifferent differentiation potentials are also defined by their source,age, preservation, culture and differentiation methods. SisakhtnezhadS., Alimoradi E., Akrami H., (2017) External factors influencingmesenchymal stem cell fate in vitro, European Journal of Cell Biology,Vol 96, Issue 1, Pages 13-33.

According to clinicaltrials.gov, there has been a significant increasein human trials using different stem cells for various applicationssince 2009. These trials, however, rely on suboptimal cellular andmatrix products either derived from mature adult people or preparedusing suboptimal non-stem cell specific methods. For example, adult bonemarrow or adipose tissue derived stem cell products cultured intwo-dimensional environments under high oxygen pressure, not normallyfound in vivo. Mendicino M. et. al., (2014) “MSC-Based ProductCharacterization for Clinical Trials: An FDA Perspective”, Cell StemCell 14, February 6.

The literature is also replete with products manufactured in ways thatalter, reduce or increase rejection risk of the product, be ittransplanted cells or matrices. For example, culturing human cord bloodstem cells in the presence of adult human bone marrow mesenchymal stemcells can elicit an adverse immunoreactivity between the cultured cellsor between the cultured cells and the host, hence reducing the desiredregenerative effect of the assumed therapeutic product. In this case,adding fetal bovine or synthetic serum to the stem cell product inculture may further reduce the optimal biology of the stem cell product.DeLima M. et. al., 2012 Cord-Blood Engraftment with Ex VivoMesenchymal-Cell Coculture, NEJM 367; 24; Gharibi B., Hughes F. J.,(2012) Effects of Medium Supplements on Proliferation, DifferentiationPotential, and In Vitro Expansion of Mesenchymal Stem Cells, Stem CellsTranslational Medicine, 1:771-782. Emnett R. J. et. al., (2016)Evaluation of Tissue Homogenization to Support the Generation ofGMP-Compliant Mesenchymal Stromal Cells from the Umbilical Cord. StemCells International. Article ID 3274054; Friedman R. et. al., (2007)Umbilical Cord Mesenchymal Stem Cells: Adjuvants for Human CellTransplantation. Biology of Blood and Marrow Transplantation13:1477-1486.

Spatial growing conditions also plays an important role in stem celltherapies. Using an ultra-thin supportive extracellular matrix as asynthetic product derived from cell cultures provides littlethree-dimensional environment to support cell growth anddifferentiation. Furthermore, a matrix derived from an animal ordifferent human is often immunologically incompatible with cells usedfor regenerative medicine. There is an extreme shortage of organ donorsand an extreme need of autologous or family related matrices and cellsfor regenerative medicine so as to avoid the complications associatedwith rejection and lifetime immunosuppression. Badylak Steven (2014)Decellularized Allogeneic and Xenogeneic Tissue as a Bioscaffold forRegenerative Medicine: Factors that Influence the Host Response. Annalsof Biomedical Engineering, Vol. 42, No. 7.

Studies showed that “mesenchymal stem cells” grown in 2D dishes in thepresence of allogeneic human umbilical cord serum show phenotypicdifferences at the structural level including smaller size, densernuclear membrane, and a reduced cytoplasm as compared to cells grown inanimal serum. Nevertheless, these same “mesenchymal stem cells” heldtheir characteristic surface marker expression (HLA-DR, CD73, CD90,CD34, CD45, CD166, and CD105), despite their differences in structure,self-renewal, and proliferative capacities. Jung J. et. al., (2009)“Mesenchymal stromal cells expanded in human allogenic cord blood serumdisplay higher self-renewal and enhanced osteogenic potential”, StemCells and Development. 18(4):559-571; Phadnis S M et. al., (2006) “Humanumbilical cord blood serum promotes growth, proliferation, as well asdifferentiation of human bone marrow-derived progenitor cells”. In VitroCellular and Developmental Biology-Animal, 42(10):283-286. Smith I. et.al., (2017) Human neural stem cell-derived cultures in three-dimensionalsubstrates form spontaneously functional neuronal networks. J Tissue EngRegen Med 2017; 11: 1022-1033.

Still another example is providing unnatural or unnecessary growthfactors to stem cells or depriving specific stem cells from growthfactors they normally need to best maintain their properties to grow,self-renew and differentiate. Cohen S B et. al., showed that cord bloodstem cells are more naïve to their environment and cord blood serumlacks a T cell activation factor. Cohen S B, Perez-Cruz I, Fallen P,Gluckman E, Madrigal J A. 1999. Analysis of the cytokine production bycord and adult blood. Hum. Immunol. 60: 331. De Waele Met. al., alsoshowed that normal cord blood CD34 positive cells, a subset of which isconsidered to contain the blood stem cell pool, have a growth factorreceptor profile and sensitivity to growth factors different than cellsfound in normal bone marrow or mobilized peripheral blood. De Waele M.et. al., (2004) Growth factor receptor profile of CD34+ cells in normalbone marrow, cord blood and mobilized peripheral blood. Eur J Haematol.March; 72(3):193-202. Hence, it is important to subject cells to theirnatural environment if one needs to control the therapy and harness theoptimal regenerative product. Sisakhtnezhad S. et. al., (2017) Externalfactors influencing mesenchymal stem cell fate in vitro. EuropeanJournal of Cell Biology 96 (2017) 13-33.

Young stem cells have a greater ability to grow and differentiate and agreater stability in culture as compared to adult mature sources of stemcells. Therefore, cord blood from neonates is expected to be a moreeffective stem cell source than bone marrow. On the other hand,umbilical cord tissue also contains cells and their secretions, isavailable in limited supply, and there can be variability betweenneonates. Sharpless N. E. and De Pinho R. A., (2007) “How stem cells ageand why this makes us grow old”, Nature Rev. Mol. Cell Biol.; Shu S. et.al., (2012), “Immunogenicity of allogeneic mesenchymal stem cells” J.Cell. Mol. Med. Vol 16, No 9, pp. 2094-2103; Lo Sardo, V. et al. (2017)Influence of donor age on induced pluripotent stem cells. Nat.Biotechnol. 35, 69-74.

Some umbilical cord tissue-derived cells are characterized and some areuncharacterized. Those cord tissue-derived cells that are characterizedtoday are defined as “mesenchymal stem cells” or MSCs usually grownusing suboptimal unnatural cell culture or harsh processing methods. Forexample, methods of isolating cells from cord tissue include the use ofnon-specific enzymes such as proteases that alter cell surface proteinsand possibly biology of the cells. Furthermore, the InternationalSociety for Cellular Therapy (ISCT) defined MSCs based on methods usinghigh oxygen pressure, incompatible serum, and a cell property thatattaches to two-dimensional cell culture environment. Consequently,living products like MSCs are defined by their suboptimal manufacturingmethods and cannot provide optimal therapeutic outcomes. In addition,cells that do not attach to conventional two-dimensional surfaces or areuncharacterized because of their small size or number are lost.Mendicino M. et. al., (2014) “MSC-Based Product Characterization forClinical Trials: An FDA Perspective”, Cell Stem Cell 14, February 6;Shuvalova N. S. et. al., (2013), “Maintenance of mesenchymal stem cellsculture due to the cells with reduced attachment rate” Biopolymers andCell. Vol. 29. N 1. P. 75-78.

In addition, for stem cells and supportive matrices to be used in tissueengineering and regenerative medicine one must consider twothings—safety and efficacy. The method of preparation of cells andtissues for transplantations is very important because manipulatingcells and tissue and introducing them to new agents, reagents andenvironments may turn these cells harmful or inefficient whentransplanted in any individual, self or not. Further, current culturemethods change stem cells in ways that can reduce or eliminate theirefficacy and compromise their safety. Indeed, cord blood transplantationprofessionals have already complained about quality of cord blood unitsthey receive from public or private banks stating that these low qualitycord blood units pose a great risk on patients receiving them. In fact,public banking standards for cord blood collection, diagnosis,processing, banking and releasing have not been established and mandateduntil October 2011. Excepting bone marrow and cord blood, all othercellular products remain in experimental stage and there are currentlyno non-blood stem cell FDA processing standards in place.fda.gov/biologicsbloodvaccines/scienceresearch/biologicsresearchareas/ucm127182.htm

Methods to collect and preserve all types of stem cells fall into twobasic categories—unmanipulated (or least minimally manipulated) andmanipulated methods.

The minimal manipulation method of collecting and freezing is mainlyused for bone marrow and cord blood. The first minimal manipulationinvolved the collection of blood and direct infusion into a patient.Alternatively, aspirates of blood or marrow were mixed with bloodanticoagulant and layered on specific density solutions likeFicoll-Hypaque to allow the density dependent separation of bloodmononuclear cells (buffy coat) from plasma and red blood cells. Themiddle “buffy coat” layer containing the blood (and other) stem cells isgently aspirated, leaving behind the top plasma layer and the bottomlayer containing red blood cells. At this stage, the buffy coat is mixedwith a cryoprotectant, like dimethyl sulfoxide (DMSO), ethylene glycolor glycerol, to a final concentration of 1% to 10% and immediately slowfrozen to −120° C. before immersing it in the liquid or gas phase of aliquid nitrogen storage tank or Dewar. A new version of this methodinvolves replacing the density solution with a set of processing bagsconnected through tubings and placed in a special device such as the AXPor SEPAX, which are automated closed systems that harvest the stemcell-rich buffy coat containing mononuclear cells from umbilical cordblood. The system reduces a unit of cord blood to a precise volumeselected by the operator and does so with high precision and safety.

In other minimal manipulation methods, tissues are excised from one areaof the body and retransplanted back in another of the same type. Forexample, skin from the leg area transplanted in an injured or burnt skinin the arm or face. Another example is scraped or shaved bone from thepelvis transplanted in a fractured or missing bone in the jaw.

The manipulated methods involve manipulating the cells longer than onehour and/or mixing the cells with agents other than water, phosphatebuffer solution, cryoprotectant and Ficoll-Hypaque. Typically, in thesemethods, mechanical sectioning and/or enzymatic digestion of tissue toseparate cells is used. Cells may be sorted, transfected for genetherapy and cultured in serum free media or media containing animal orgenetically non-identical human sera, or genetically non-identicalplatelet lysates. Growth factors like epidermal growth factor andhormones like insulin may also be added to stimulate growth andproliferation of cultured cells. Furthermore, cells may be cultured in atwo-dimensional or three-dimensional matrix where they may be guided togrow into a specific form. Alternatively, a piece of extracted tissuemay be decellularized to create a matrix in which autologous orallogeneic cells may be infused. The matrix containing the necessarycells can be transplanted back into a patient to regrow and heal adegenerated tissue. These methods have a higher risk of negativelyimpacting stem cell safety and efficacy. Shakouri-Motlagh A. et. al.,(2017) Native and solubilized decellularized extracellular matrix: Acritical assessment of their potential for improving the expansion ofmesenchymal stem cells. Acta Biomaterialia 55 (2017) 1-12; Meral Beksac(2016) How to Improve Cord Blood Transplantation. By Enhancing CellCount or Engraftment? Frontiers in Medicine, Vol. 3, Article 20; GentileP. et. al., (2017) Concise Review: The Use of Adipose-Derived StromalVascular Fraction Cells and Platelet Rich Plasma in Regenerative PlasticSurgery. Stem Cells; 35:117-134; Smith I. et. al., (2017) Human neuralstem cell-derived cultures in three-dimensional substrates formspontaneously functional neuronal networks. J Tissue Eng Regen Med 2017;11: 1022-1033; Sisakhtnezhad S. et. al., (2017) External factorsinfluencing mesenchymal stem cell fate in vitro. European Journal ofCell Biology 96 (2017) 13-33.

What is needed in the art are better methods isolating, culturing andpreserving stem cells and compatible biomatrices that provides morereliable, reproducible, safer and efficacious products.

BRIEF SUMMARY OF THE INVENTION

The present disclosure describes a method of collection and a four wayprocessing of human umbilical cord to manufacture four products: 1)Autologous or HLA- and optionally gender-matched plasma and red bloodcell-reduced cord blood for transplantation in an autologous, related orunrelated patient; 2) Autologous or HLA- and optionally gender matchedserum, plasma, or platelet lysate, for use in culturing cells; 3) Acomplete mixture of autologous or HLA- and optionally gender-matchedcells liberated from a portion of intact cord tissue; 4) An autologousor HLA- and optionally gender-matched decellularized tissue matrixprepared from an intact portion of cord tissue, which can be cut intodesired shapes or left as a slurry of small matrix fragments; 5) Acombination of 3 and 4, wherein the mixture of cells is reinfused backinto the decellularized tissue matrix. Any combination of products 1-5is possible, and each of the products can be used as is, stored by e.g.,freezing, or cultured to amplify cells, or any combination therein.

In addition, methods of producing stem cell products for research andtherapies are provided. For example, a complete cell mixture liberatedfrom intact cord tissue can be infused back into the decellularizedtissue matrix and subsequently cultured in three dimensions (3D) inmedia containing autologous serum, plasma or platelet lysate in thepresence of low oxygen tension. This methodology produces well-preservedcell and biomatrix products with better regenerative potential forinjured, diseased and aging patients or animals.

In more detail, the disclosure is a method of producing stem cells andtissue matrix. Preferably, the tissue preparation commences within 48hours, preferably within 16 or 12 hours, of obtaining the tissue, andthe process occurs in at least a class 10,000 clean room facility.

The method begins with obtaining (directly or indirectly) cord tissuefrom a newborn animal. The tissue will be sterilized then cut into twoor more portions. A portion of the tissue is used to preparedecellularized matrix from whole cord tissue that can function as athree dimensional matrix for subsequent cell culturing or tissueengineering techniques. Another portion of the same whole cord tissue isused to collect all cells within this whole cord tissue.

For decellularized matrix, the tissue is used as whole or cut intodesired fragments shapes before being chemically, enzymatically and/ormechanically treated to liberate cells or cellular debris, leaving adecellularized tissue matrix from which at least 70%, preferably 75%,80%, 85%, 90% or 95%, of the cells have been removed. Although anymethod of decellularization can be used, we prefer methods that avoidthe use of enzymes or harsh chemicals.

It is usually preferred that the vessels of the tissue not be separatedfrom the rest of the tissue, because the tissue with vessels may beimportant and necessary for tissue engineering and properthree-dimensional cell culturing conditions.

For decellularized matrix, the tissue can be shaped beforedecellularization, after decellularization, or both, butdecellularization is easier with greater surface area, so it ispreferred that the tissue is somewhat cut or shaped beforedecellularization. A piece of cord tissue can thus be minced, or cutinto flat slices or blocks, or spiral cut, or any other desired way soas to create a gel or slurry of small fragments, sheets, blocks, orrolls of material having different thicknesses, lengths and widthsranging from micrometers to meters. These shapes are chosen according tothe intended use of the decellularized matrix material, e.g., a largesheet being suitable for burned skin replacement.

For decellularized matrix, it may be preferred that the fragmentation orcutting is made on cord tissue that is fresh, but it could also beembedded in a biodegradable natural or synthetic polymer or even frozenusing for example a fixed or moving blade or laser. Embedding orfreezing the cord will aid in e.g., spiral slicing of cord tissue.

A second portion of the tissue is used to obtain all the cells insidethe tissue by mechanically dissociating the tissues from the cells. Thetissue is gently mechanically dissociated, e.g., with a blade, a laseror homogenizer. Then the tissue fragments are gently agitated to releaseintact cells. Although any method of liberating cells can be used, weprefer gentle agitation and other methods that avoid the use of enzymes.

The liberated cells will be a complete mixture of all cell types foundin the tissue, including, endothelial cells, epithelial cells,mesenchymal cells, adherent and non-adherent cells, mesenchymal stemcells, mesenchymal progenitor cells and others. The goal is to liberate,collect and preserve all cell types in the tissue.

The cells are then cultured and/or directly stored, either alone or withautologous serum and/or decellularized tissue. Cells can also becultured alone or with autologous serum and/or decellularized tissue.Preferably, they are both stored and cultured with autologous serum,etc., as this provides a better product. Also, preferred, they arecultured with decellularized tissue at some point before use, as thedecellularized tissue provides a natural matrix for 3D growth.

Culturing is preferably via a three dimensional cell culture in ahypoxic environment at 37° C. in a medium supplemented with autologousor syngeneic serum, plasma or platelet lysate. Preferably, the culturingstep is in a humidified carbon dioxide cell culture chamber. The pointof culturing the cells in a hypoxic environment is to enhance stem cellself-renewal and differentiation potential. Hypoxic environment and 3Dculturing provides a more natural environment, and minimizes thecellular changes that can occur on culturing. Further, the addition ofdecellularized tissue fragments or shaped decellularized tissue matrixprovides an example of 3D environment that more closely mimics the invivo environment.

The starting tissue can be any tissue from the body that contains somestem cells, and preferably includes skin, umbilical cord, heart, brain,or hair. However, body fluids such as urine, blood and the like can alsobe used. A preferred tissue is whole umbilical cord tissue, which cancontain more or less cord blood.

The decellularized tissue matrix, tissue fragments and the cells can beused immediately or stored for future use, and they can be stored eithertogether or separately, again depending on the ultimate use. Storage canbe by freezing, e.g., with a cell protecting agent such as DMSO orglycerol, but the tissue matrix can be freeze dried or lyophilized aswell, and rehydrated for use according to known techniques.

Importantly, the method produces a mixture of differentiated cells, stemcells and progenitor cells. These cells can be used as is, or used afterfirst re-infusing back into a decellularized tissue matrix, prepared asdescribed herein. The stem cells can also undergo further separation,amplification and differentiation into a desired cell type, but wespecifically contemplate using a mixture of cells in therapeuticapplications. The mixture of cells provides a more natural environment,where stem cells can respond to signals from other cells and/or thematrix to stimulate differentiation into the desired cell type.

When used together with a decellularized tissue matrix, that matrix canbe from the same or a genetically identical (syngeneic) animal, but itcould also be from a different animal, since the matrix is largelydecellularized and the matrix itself will be minimally antigenic. Thedecellularized tissue matrix provides the cells with a scaffold forgrowth, as well as the needed growth factors and the like, ensuring ahighly safe and effective, biocompatible tissue replacement.

Another embodiment is a method of cell therapy, wherein the productsdescribed herein are transplanted into a patient. Other embodiments ofthe disclosure are methods of stem cell therapy, using one or more ofthe products and/or methods described herein.

In more detail, the invention includes any one or more of the followingembodiments, in any combination(s) thereof:

A method of producing stem cells and tissue matrix, comprising: a)obtaining a tissue from an animal source; b) cleaning said tissue with asterilizing agent; c) mechanically dissociating said tissue to producecells and tissue fragments; d) mechanically or chemically treating saidtissue fragments to produce cells or lysed cells and tissue matrix; e)culturing said cells in a three dimensional cell culture in hypoxicoxygen environment at 37° C. in a medium supplemented with serum, plasmaor platelet lysate isolated from said animal source or a geneticallyidentical animal source, wherein said cells are cultured alone ortogether with said tissue matrix or said tissue fragments. Any methodherein described, whereby tissue is selected from skin, umbilical cord,heart, brain, or hair. Any method herein described, whereby i) thetissue matrix and the cells are cultured together and stored together,ii) the tissue matrix and the cells are cultured together and storedseparately, iii) the tissue matrix and the cells are cultured separatelyand stored separately, iv) the tissue fragments and the cells arecultured separately and stored separately; v) the tissue fragments andthe cells are cultured together and stored together or vi) the tissuefragments and the cells are cultured together and stored separately. Anymethod herein described, whereby said cells are a mixture ofdifferentiated cells, stem cells and progenitor cells. Any method hereindescribed, whereby steps c-e) commence within 48 hours of said obtainingstep. Any method herein described, wherein said mechanicallydissociating step is by slicing or dicing said tissue with a blade. Anymethod herein described, wherein said tissue is sliced into a desiredshape. Any method herein described, where said culturing step is in ahumidified carbon dioxide cell culture chamber. Any method hereindescribed, where said tissue matrix and cells are stored together orseparately at temperatures between −80° C. and −196° C. Any methodherein described, where said tissue matrix is freeze-dried and stored at−80° C. Any method herein described, where said freeze-dried tissuematrix is thawed in the presence of phosphate buffer solution or water.Any method herein described, where said tissue matrix is lyophilized andstored at room temperature. Any method herein described, where saidlyophilized tissue matrix is reconstituted with water or phosphatebuffer solution. Any method herein described, further comprisingreintroducing said cells into said tissue matrix before use. Any methodherein described, wherein said cells and said tissue matrix areseparated before storage and further comprising reintroducing said cellsinto a genetically identical or different tissue matrix before use. Amethod of producing stem cells comprising: a) obtaining an umbilicalcord from a source; b) cleaning said umbilical cord with a sterilizingagent; c) mechanically dissociating said umbilical cord to producetissue fragments and cells; d) culturing said tissue fragments and saidcells in a three-dimensional cell culture in a hypoxic oxygenenvironment at 37° C. in a medium; and e) collecting said cells and saidtissue fragments, wherein said cells are a mixture of differentiatedcells, stem cells, and progenitor cells. Any method herein described,wherein said medium is supplemented with serum, plasma or plateletlysate isolated from said source or a genetically identical source. Anymethod herein described, wherein said mechanically dissociating iscutting thin slices. Any method herein described, wherein said cells arereinfused into a tissue matrix before use. Any method herein described,wherein said tissue matrix is from said source. Any method hereindescribed, wherein said tissue matrix is from a different source. Amethod of preparing a cell mixture of all cell types present inumbilical cord tissue, said cell mixture obtained by the followingsteps: a) obtaining a first intact portion of umbilical cord tissue froman umbilical cord of a first newborn animal; b) cleaning said firstintact portion of umbilical cord tissue with a sterilizing agent; c)mechanically dissociating said first intact portion of umbilical cordtissue into smaller fragments; d) mechanically liberating cells fromsaid smaller fragments; and e) collecting a cell mixture of all cellstypes liberated from said smaller fragments, wherein said cell mixtureincludes differentiated cells, stem cells and progenitor cells; and f)combining said cell mixture with autologous or syngeneic or HLA- andgender matched serum, plasma or platelet lysate for culturing orstorage, or both. A method of making biomatrix comprising: a) obtaininga second intact portion of umbilical cord tissue from said first newbornanimal or from a second newborn animal; b) cutting said second intactportion of umbilical cord tissue to a desired shape without separatingvessels from the rest of the cord tissue; c) decellularizing said secondumbilical cord tissue to produce a decellularized tissue; d) culturingsaid cell mixture together with said decellularized tissue such thatsaid decellularized tissue provides a 3D scaffold for 3D culturing ofsaid cells mixture, wherein said 3D culturing is in a 0.1% to 5% or0.5-7% oxygen environment at 37° C. in a medium supplemented withautologous or syngeneic or HLA- and gender-matched serum, plasma orplatelet lysate. Any method herein described, wherein said cell mixturecomprises about 10% of non-adhering pluripotent cells, 40% of adheringpluripotent stem cells, and 50% of differentiated cells, wherein cellsin the cell mixture having sizes ranging from 3-100 μm in diameter. Anymethod herein described, wherein said decellularized tissue is from saidfirst newborn animal. Any method herein described, further comprisingadding mononuclear cells obtained from the umbilical cord blood fromsaid first newborn animal to the cell mixture being cultured. Any methodherein described, wherein said culturing is under 0.5% to 7% oxygen. Anymethod herein described, wherein said medium is supplemented withumbilical cord plasma obtained from umbilical cord blood from the firstnewborn animal. Any method herein described, further comprising addingmononuclear cells obtained from the umbilical cord blood from the firstnewborn animal to said culture. Any method herein described, whereinsaid culturing is under 0.5% to 7% oxygen. Any method herein described,further comprising adding mononuclear cells obtained from the umbilicalcord blood from the first newborn animal to said cell mixture. Anymethod herein described, wherein said cell mixture is cultured under0.5% to 7% oxygen. Any method herein described, wherein the cell mixturebeing cultured under 0.5% to 7% oxygen in a medium supplemented withumbilical cord plasma obtained from umbilical cord blood from the firstnewborn animal. A decellularized tissue biomatrix, said biomatrix beingobtained by the following steps: a) obtaining a first intact portion ofumbilical cord tissue from the umbilical cord of a first newborn animal;b) cleaning said first intact portion of umbilical cord tissue with asterilizing agent; c) cutting said first intact portion of umbilicalcord tissue to a desired shape without separating vessels from the restof the cord tissue and decellularizing said first intact portion ofumbilical cord tissue to produce decellularized tissue; and d)optionally further mechanically changing the size or shape or both ofthe decellularized tissue to obtain said biomatrix. Any method hereindescribed, wherein the biomatrix is combined with a mixture of cellsisolated from a second intact portion of cord tissue from said newbornanimal or a second newborn animal Any method herein described, whereinsaid combination is cultured under 0.5% to 7% oxygen. Any method hereindescribed, wherein said combination is cultured under 0.5% to 7% oxygenin a medium supplemented with umbilical cord plasma obtained fromumbilical cord blood from said first newborn animal. Any method hereindescribed, wherein said combination is cultured under 0.5% to 7% oxygenin a medium supplemented with autologous, syngeneic or HLA matchedplasma, serum or platelet lysate. Any method herein described, saidbiomatrix being stored at temperatures between −80° C. and −196° C. Anymethod herein described, said biomatrix being lyophilized and stored at−80° C. Any method herein described, said biomatrix being lyophilizedand stored at room temperature. Any method herein described, where thelyophilized acellular biomatrix is reconstituted with water or phosphatebuffer solution. Any method herein described, where the lyophilizedacellular biomatrix is reconstituted with water or phosphate buffersolution. Any product herein described, wherein one or more of i) saidcell mixture or ii) said serum, plasma or platelet lysate or iii) saiddecellularized tissue is a pooled HLA-matched product, each pooledportion of said HLA-matched product having the same at least 3 matchedMHC loci. A method of culturing stem cells, said method comprising: a)obtaining stem cells from a first donor; b) obtaining sera from an HLA-and gender matched second donor having the same gender and at least 3HLA types that are the same as said first donor; and c) culturing saidstems cells in a medium supplemented with 1-10% of said sera. A methodof culturing stem cells, said method comprising: a) obtaining stemcells; b) obtaining plasma, serum or platelet lysate that is HLA-matchedand optionally gender matched to said stem cells and having at least 3MHC loci that are matched to said stem cells; and c) culturing saidstems cells under hypoxic (0.1-7% O₂) conditions in a mediumsupplemented with 1- 10% of said plasma, serum or platelet lysate A stemcell product for use in a stem cell treatment of a patient, said productcomprising a cell mixture of all cell types cells from umbilical cordtissue that is cultured in a 3D scaffold made from an intact portion ofdecellularized cord tissue under hypoxic conditions in a mediasupplemented with 1- 10% serum, plasma or platelet lysate, wherein saidcell mixture and said serum, plasma or platelet lysate are from a samedonor and where said donor has at least 3 HLA matches with a patient tobe treated with said product. Preferably, the decellularized tissuematrix is also from the same source, although this is not essential. Astem cell product for use in a stem cell treatment of a patient, saidproduct comprising a cell mixture of all cell types cells mechanicallyfrom comminuted umbilical cord tissue that is cultured in a 3D scaffoldmade from an intact portion of decellularized cord tissue under hypoxicconditions in a media supplemented with 1-10% serum, plasma or plateletlysate, wherein said cell mixture and said serum, plasma or plateletlysate are from a same donor and where said donor has at least 3 MHCloci matched with a patient to be treated with said product. Mostpreferred, the donor IS the patient. Preferably, the decellularizedtissue matrix is also from the same source, although this is notessential. A regenerative medicine product for use in treatment of apatient, said product comprising a cell mixture of cell types liberatedfrom umbilical cord tissue, wherein said cell mixture is cultured in a3D scaffold made from an intact portion of decellularized cord tissueunder hypoxic conditions in a media supplemented with 1-10% serum,plasma or platelet lysate, wherein said cell mixture, decellularizedcord tissue and said serum, plasma or platelet lysate have the same atleast 3 MHC loci matched with a patient to be treated with said product.A stem cell product for use in a stem cell treatment of a patient, saidproduct comprising a cell mixture of cell types mechanically releasedfrom comminuted umbilical cord tissue that is cultured in a 3D scaffoldmade from an intact portion of decellularized cord tissue under hypoxicconditions in a media supplemented with 1-10% serum, plasma or plateletlysate, wherein said cell mixture, said decellularized cord tissue andsaid serum, plasma or platelet lysate each have the same at least 3 MHCloci matched with a patient to be treated with said product. Preferably,all combination products here come from the same source and are thusautologous. However, where material is limiting, any one or more of thevarious components can be HLA matched, so long as the same 3 HLAmatching loci are present in all heterologous components. Ideally, wherecomponents are pooled, the same donors provide all pooled portions, thusminimizing the variability in products. A product comprising mixed cellson a biomatrix scaffold, each as described herein, that is 3Dhypoxically cultured with serum, plasma or platelet lysate, as describedherein. Such products can be further combined with mononuclear cellsfrom cord blood or with other products.

There are substantial differences between the disclosure as describedand claimed herein and the prior art methodologies. These are elaboratedon below.

The facility in which the procedure is executed should be a dedicatedclean room with positive air-flow. The clean room facility is made of atleast three zones, the actual clean room where processing occurs(preferably class 10,000 (Iso 4) or better), as well as a gowning room(preferably class 10,000 or better), and an entry airlock room(preferably class 10,000 or better). Preferably, the facility also hassample receiving rooms, diagnostic rooms (for performing tests forinfectious disease, and the like) and final sample storage rooms, aswell as pass-through hatches for samples to pass to each next processingzone.

By “class 10,000 compliant,” what is meant that the facility at leastcomplies with the US FED STD 209E regulations or equivalent. Also,preferably the facility is a dual process facility, with separate butmirrored facilities for public and private stem cell processing andbanking facilities. A preferred facility is described in U.S. Pat. No.8,656,670, filed Jul. 22, 2012, and incorporated herein by reference inits entirety.

Following expectant mother screening and her consent, cord blood iscollected aseptically after the baby is born vaginally or by C-sectionand separated from the mother. While the placenta is still in utero orremoved, cord blood is collected using a needle inserted in theumbilical vein that allows blood to drain into a bag containinganti-coagulant like Citrate Phosphate Dextrose (CPD), EDTA or heparin.

Following blood collection, whole cord tissue is collected, rinsed fromblood and sterilized before it is placed in another collection bag.Blood and tissue bags are each doubly wrapped and placed in differenttemperature controlled compartments of a shipping container, sealed andimmediately shipped to our hybrid tissue bank for diagnostics,processing and banking.

In a clean room, cord blood is transferred to an AXP® (CescaTherapeutics) or SEPAX® (Biosafe) processing bag, then the blood isdifferentially centrifuged to separate it into three products: a plasmaand red blood cell reduced cord blood bag, a plasma bag and a red bloodcell bag. Then, each of plasma, red blood cell and blood stem cell bagsare separated. Cord blood plasma bag is aliquoted into different steriletubes or vials wrapped and saved at temperatures between −196° C. and 4°C.

The blood stem cell bag is infused with a cryoprotectant like DMSO,ethylene or propylene glycol and an additive, then slowly or snap frozento −196° C. and banked in 24/7 monitored dewars. On the other hand,whole cord tissue is transported at 0° C. to 8° C. to our hybrid tissuebank. The whole cord is cut into two or more pieces. Some of the piecesare used to collect all cells from the tissue of the cord. The rest ofcord pieces are used to produce a decellularized cord tissue matrix.

From each cord the following individual products are isolated: i) cordblood, ii) cord blood plasma and related products, iii) cord tissuederived cells and iv) cord tissue derived decellularized tissue matrix,and v) combinations thereof.

The method uses the whole tissue to produce cell mixtures anddecellularized tissue matrix, after emptying cord tissue from cord bloodand collecting the blood in a bag, although of course cord tissue maycontain some amount of remaining blood.

The tissue is CUT to a certain shape (without separating vessels fromrest of tissue) before or after (or both) producing a decellularizedtissue matrix of the desired shape, devoid of cells, with or preferablywithout using nucleases or other enzymes, as described in US20050203636,U.S. Pat. Nos. 7,775,965, and 7,318,998.

In the initial isolation and freezing or culturing, the method is notfocused on specific cell types (such as mesenchymal stem cells orendothelial stem cells), nor on using 2D cell cultures and fetal bovineserum supplementation, as described in US20110129918, US20060223177.Instead, the cells are isolated as a mixture of different cell types andfrozen or cultured in 3D as a suspension or on 3D surfaces, preferably a3D decellularized tissue matrix derived from the same or anothernewborn, again providing a more natural culture environment.

Preferably, we use autologous or syngeneic serum and other products,prepared from the cord blood and also from blood donated by the sameindividual later in life. The cord blood is only present in smallquantities, and thus has previously not been used in this way. However,we have discovered that there is enough cord blood for an initial shortculture and/or storage of the cord cell mixture, and when the cells arelater needed for treatment, the individual can provide additional bloodfor further culture.

Serum and platelets contain natural amounts of growth factors andcytokines necessary to maintain cells and stem cells in their naturalcross talk environment being either an undifferentiated state or a stateof response to differentiation factors. In addition to making theproduct more potent, using autologous or syngeneic blood and tissuecomponents and reducing foreign agents in cell culture media duringbiological preparations like stem cell processing is more accepted bythe scientific community. More importantly it may eventually reduce thetime to market these newborn derived products by facilitating FDAapproval and hence patients may benefit more quickly.

As another option, given the small amount of cord blood available(Wagner J E. et. al., (2002) “Transplantation of unrelated donorumbilical cord blood in 102 patients with malignant and nonmalignantdiseases: influence of CD34 cell dose and HLA disparity ontreatment-related mortality and survival”. Blood, 100:1611-1618), HLAtyped blood and cells could be used, including pooled products with atleast 3 MHC loci that are the same, and that where pooled products areused they each have the same matched loci. The human leukocyte antigen(HLA) system or complex is a gene complex encoding the majorhistocompatibility complex (MHC) proteins in humans. These cell surfaceproteins are responsible for the regulation of the immune system inhumans. HLA genes are highly polymorphic, which means that they havemany different alleles, allowing them to fine-tune the adaptive immunesystem.

Given the small amount of cord tissue cells and decellularized matrixavailable, we also envisage using cord blood, cord tissue-derived cellsand cord tissue-derived decellularized cord tissue matrix that arematched at HLA loci and optionally gender matched to help transplantpatients who do not have their own normal cord blood and cord tissuesaved. Specifically, we can produce one or more combinations of cordblood, cord tissue cells, cord tissue matrix and cord blood sera, plasmaand platelets lysates where one or more of these products are derivedfrom the same HLA- and optionally sex matched donors, such that thepooled and combination products all have the same at least 3 matched MHCloci. Although such HLA-matched products are ideally taken fromHLA-matched cords, they can also be taken from HLA-matched adults.

We also allow pooling of cord blood products, cord tissue cell products,decellularized matrix products and combinations thereof, but each of thedonor sources should have same at least 3 matched MHC loci, so that allpooled and combination products ideally all have the same 3 MHC loci,which also matches to patient in which the various products are to beused. Combination products can also include combinations of autologousand these HLA matched components.

The purpose of this matching is to minimize immuno-reactivity betweenthe cells, matrix and serum/plasma/platelets products when culturedtogether. It also minimizes rejection and improves transplant outcomewhen these products are transplanted in an HLA-matched patient. Hence,unlike current practice, a pool of allogeneic normal cord blood, sera orplasma, cord tissue cells and cord tissue matrix that are genderspecific and/or matched at HLA loci will significantly help transplantpatients who do not have their own normal cord blood, serum or plasma,cord tissue cells and cord matrix saved. McNiece I. et. al., (2004) “Exvivo expansion of umbilical cord blood hemopoietic stem and progenitorcells”. Exp Hematol., 32:409-413.

The cells are grown in 3D (e.g., in a mini bioreactor). Threedimensional culture techniques have been applied to stem cell culturing.However, culturing or co-culturing cells in a bioreactor, in or on athree dimensional surface derived from the same or similar geneticsource is novel and is expected to improved biological properties. Whenit comes to stem cells, it is important to preserve theirdifferentiation potential in response to stimuli. 3D culturing onmatrices or scaffold derived from the same cell source is expected toenhance the usefulness of the cells when transplanted alone or whentransplanted in combination with the matrix or scaffold. Thetransplanted matrix or scaffold provides cells with a surface that isappropriate for their migration, proliferation or differentiation on orwithin the matrix.

Stem cells are also cultured in a hypoxic environment. Currentlytransplanted cells are cultured under atmospheric oxygen pressure.Herein, by contrast, we use cells that are gradually or suddenly exposedto a hypoxic environment ranging from 0.5% to 7% oxygen while culturedin or on a 3D matrix in the presence or absence of 1% to 10% autologousblood components in an incubator at 37° C. for e.g., 1-7 days, or up to2 or 3 weeks, or more. These conditions produce a novel biologicallyactive cellular product that is more useful for regenerative medicine.

The cell mixture liberated from cord tissue is reinfused into theacellular tissue matrix. Cells are reinfused at a specific density of50,000 to 600,000 cells per cubic centimeter of a piece of vascularizedor avascularized tissue that has been decellularized. For cellcombinations, for example a 5 to 1 ratio of mesenchymal stem cells toendothelial progenitor cells are infused in a decellularized piece ofcord tissue and incubated under conditions mentioned above.Alternatively, stem cells can be stimulated to differentiate into adesired cell type, like a muscle, cartilage, bone or skin cell, prior toinfusing them at a specific density of 50,000 to 10⁷ cells per cubiccentimeter of decellularized matrix.

By “animal” herein what is meant is any non-human animal or human.Preferably the animals are mammals.

By “medium” herein what is meant is phosphate buffer saline (PBS) orchemically defined medium.

By “decellularized cord tissue-derived matrix” herein what is meant isthat at least 70% of the original cells of said tissue have beenremoved, leaving behind a native extracellular matrix (ECM) containingconnective tissue, proteoglycans, such as heparin sulfate, chondroitinsulfate, and keratin sulfate, non-proteoglycan polysaccharides, such ashyaluronic acid, fibers such as collagen and elastin, and miscellaneouscomponents such as fibronectin, laminin and vitronectin. This product isnot the same as ECM that is secreted by cells grown in culture, but hasa more natural structure since it originated from natural tissue, ratherthan by cells grown in culture.

By “hypoxic” what is meant herein is that the cells are cultured in lowoxygen tension with less than usual pressure of 159 mm Hg (21% O₂), butare not anoxic. Preferably, 0.1-15% O₂ is used, preferably or 0.5-7% or0.5-5%, or 1-5% O₂ for cells from the umbilical cord.

By “mechanically dissociating” what is meant is physical method ofreducing tissue size, e.g., by slicing or cutting, triturating orhomogenizing, mortar and pestle, and the like. Preferably, the tissue iscut into fine strips or cubes with a blade.

By “mechanically liberating” what is meant are physical methods ofgently liberating cells from tissue, such as gentle homogenization orrocking on a rocker plate or passing a slow flow over the tissue, andthe like.

By “HLA-matched” what is meant is cells or blood products are fromHLA-matched donors, wherein at least three (3) major histocompatibilitycomplex (MHC) genes match. When there is 100% genetic matching of ALLMHC genes, the materials are autologous. Pooled and combination productsfrom pooled resources should also have the same 3 HLA matched loci.

By “matched” here, we refer to the recipient as the genome against whichloci are matched. Thus, HLA-matched products must have at least 3 MHCloci that match the patient where the product is to be used.

As used herein, “intact portion” means the cord tissue is used as is,and the vessels are not dissected out for separate use, although thecord itself can be halved or cut into thirds, or comminuted, etc.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims or the specification means one or more thanone, unless the context dictates otherwise.

The term “about” means the stated value plus or minus the margin oferror of measurement or plus or minus 10% if no method of measurement isindicated.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or if thealternatives are mutually exclusive.

The terms “comprise,” “have,” “include” and “contain” (and theirvariants) are open-ended linking verbs and allow the addition of otherelements when used in a claim.

The phrase “consisting of” is a closed linking verb and does not allowthe addition of any other elements.

The phrase “consisting essentially of” occupies a middle ground,allowing the addition of non-material elements such as extra washes,precipitations, drying or various buffers and the like.

The following abbreviations are used herein:

GMP Good Manufacturing Practices FDA Food and Drug Administration DMSODimethylsulfoxide DMEM Dulbecco Modified Essential Medium SCR Stem CellReserve PAA Peracetic Acid

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows some of the products derived from each umbilical cord andtheir use singly or in combination with other products derived from thesame umbilical cord.

FIG. 2 is a schematic diagram illustrating possible combination of cordblood cells, cord blood serum/plasma or platelets lysate, cord tissuecells and cord matrices products, singly or combination thereof to treata degenerated tissue.

FIG. 3 is a schematic diagram illustrating the development process ofcells.

FIG. 4A-I are illustrations of mechanical dissociation of umbilical cordtissues to produce decellularized matrices. This figure shows how anintact whole umbilical cord devoid of blood is sliced, cut or comminutedbefore or after decellularization.

FIG. 5 is another illustration of products derived from each umbilicalcord.

FIG. 6 Describes the process of collecting and preparing cells from aportion of the cord for freezing or 3D culturing.

FIG. 7 Describes the process of pooling cord cells and cord tissues frommore than one donors.

TABLE 1 shows the possible combination of 100% genetically compatibleserum, plasma or platelet lysate as well as cord blood and cordtissue-derived cells and matrix from newborn A before or after culturingat 37° C. in low oxygen tension and before using for research ortransplanting in same patient A, or an HLA- and optionally sex-matchedpatient.

TABLE 2 shows the possible combination of 100% genetically compatibleserum, plasma or platelet lysate as well as cord blood and cordtissue-derived cells from newborn A with or without a pooled HLA- andoptionally sex-matched cord tissue-derived matrix from a differentnewborn before or after culturing at 37° C. in low oxygen tension andbefore using for research or transplanting in same patient A or an HLA-and optionally sex-matched patient.

TABLE 3 shows the possible combination of 100% genetically compatiblecord blood and cord tissue-derived cells and matrix from newborn Acultured in the presence or absence of pooled HLA- and optionallysex-matched blood sera, plasmas or platelet lysates before or afterculturing at 37° C. in low oxygen tension and before using for researchor transplanting in same patient A or an HLA- and optionally sex-matchedpatient.

TABLE 4 shows the possible combination of 100% genetically compatiblecord blood and cord tissue-derived cells from newborn A cultured in thepresence or absence of pooled HLA- and optionally sex-matched bloodsera, plasmas or platelet lysates with or without pooled HLA- andoptionally sex-matched cord tissue-derived matrix from the same pool ofdonors before or after culturing at 37° C. in low oxygen tension andbefore using for research or transplanting in same patient A, or an HLA-and optionally sex-matched patient.

TABLE 5 lists various unlimiting embodiments of the invention, showingthe possible mix of products of HLA- and optionally sex matched cordblood units, HLA- and optionally sex matched cord blood units cordtissue-derived cells, HLA- and optionally sex matched cord blood unitsblood sera, plasmas or platelet lysates and HLA- and optionally sexmatched cord blood units cord tissue-derived matrices all derived fromthe same pool of donors before or after 3D culture at 37° C. in lowoxygen tension and delivery to research or transplanting in an HLA- andoptionally sex matched patient.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides any one or more of the followingembodiments, in any combination: 1) 3D culture expanded cord blood inthe presence of autologous cord tissue-derived cells and plasma; 2) 3Dculture expanded cord blood in the presence of autologous cordtissue-derived matrix and plasma; 3) 3D culture expanded cord blood inthe presence of autologous cord tissue-derived cells, cordtissue-derived matrix and plasma; 4) cord blood sera and other productsfor use in storage and culturing of cells; 5) umbilical cordtissue-derived biomatrix, which is decellularized cord tissue withintact or broken vessels; 6) umbilical cord tissue-derived cells, whichis a complete mixture of cells mechanically liberated from the cordtissue; 7) a mixture of 5 and 6, where the mix of cord tissue cells arereinfused back into the decellularized tissue, 8) a mixture of cordblood mixed with autologous 5 and 6, where the mix of cord tissue cellsare reinfused back into the decellularized tissue.

In any of the above, the cells can be amplified in culture before use,and the amplification can occur before storage or after storage (orboth), and occur before combining the matrix with the cell or after suchcombination, or combinations thereof. Preferably, the cells are bothstored and cultured with autologous or syngeneic serum and similarproducts, although HLA and gender matched serum and similar products canbe used as well.

Furthermore, the decellularized cord tissue can be used with any otherstem cells, and are not limited to use with the cord tissue mixture ofcells. Thus, they can be used with stem cells from cord blood, mixedcells from cord blood, adult stem cells from bone marrow, epithelia,adipose tissue, and the like. As yet another alternative, the mixture ofcells can be combined with other cell types for use.

Additional products may comprise the following, as seen in Tables 1through 5: umbilical cord blood cells and umbilical cord tissue-derivedcells, preserved hypoxia conditioned whole umbilical cord tissue-derivedmatrix cultured in 0.5%-7% or 0.5%-5% oxygen tension in the presence orabsence of 100% genetically compatible cord blood plasma, 100%compatible umbilical cord tissue-derived matrix and cells cultured in0.5%-7% oxygen tension in the presence of 100% genetically compatiblecord blood plasma; 100% compatible umbilical cord tissue-derived matrixand umbilical cord blood mononuclear cells cultured in 0.5%-7% oxygentension in the presence of 100% genetically compatible cord bloodplasma, 100% compatible umbilical cord tissue-derived matrix and cellsand umbilical cord blood mononuclear cells cultured in 0.5%-7% oxygentension in the presence of 100% compatible cord blood plasma, 100%compatible umbilical cord blood mononuclear cells and umbilical cordtissue-derived cells cultured in 0.5%-7% oxygen tension in the presenceor absence of cord blood plasma, and umbilical cord tissue-derived cellscultured in 0.5%-7% oxygen tension in the presence or absence of cordblood plasma. The products of this disclosure also include pooled HLA-and optionally sex matched cord tissue-derived cells, pooled HLA- andoptionally sex matched sera, plasmas or platelet lysates obtained frompooled HLA- and optionally sex-matched blood units, HLA- and optionallysex matched whole umbilical cord tissue matrix obtained from pooled HLA-and optionally sex-matched umbilical cord tissues and the combination ofthese products before and after 3D culturing.

Cord blood and cord tissue are collected and the cord blood separatedfrom the tissue as much as possible. The cord blood is typicallycollected in anticoagulant supplemented bag then mononuclear cells orbuffy coat are separated as much as possible by centrifugation from redblood cells and plasma using common blood processing techniques likeFicoll Hypaque density gradient centrifugation or the “closed technique”using AXP AutoExpress from Cesca Therapeutics or Sepax from Biosafe.Consequently, cord blood products are a bag of cord blood containingmononuclear cells made of hematopoietic lineage cells such aslymphocytes, monocytes, stem and progenitor cells as well as mesenchymalstromal cells.

Cord blood processing also produces a bag of red blood cell concentrateand another bag of plasma. Serum can also be collected from coagulatedcord blood that is centrifuged at 200-800 g for 5-20 min to sediment allcells. Serum is blood without fibrinogen. It contains salt, water,antibodies, non-clotting proteins like growth factors and antigensalthough some clotting proteins remain. It is used right away forculturing cells or immunotherapy or for cryostoring biological products.Plasma is the liquid constituent of blood when it is not coagulated. Itcontains salt, water, antibodies, proteins, clotting factors andantigens. Plasma is derived from anticoagulated blood that iscentrifuged at 200-800 g for 5-20 min to remove blood cells. Plasma alsohas a higher level of proteins than serum because during serummanufacturing some proteins get lost during coagulation andsedimentation. Plasma is useful as a source of growth factors for cellculture and for cell and tissue storage. It is also useful to treatpeople suffering from burns, shock, trauma, and other medicalemergencies. The proteins and antibodies in plasma are also used tocreate therapies for rare chronic conditions, such as autoimmunedisorders and hemophilia.

Platelets are non-nucleated fragments of mature megakaryocytes. Thereare several centrifugation protocols to collect platelets fromanticoagulated blood. One example is to spin anticoagulated blood at 300g for 5 min at 12° C. followed by collecting the supernatant andspinning at 700 g for 17 min. The resulting pellet contains plateletsand some red and white blood cell impurities. Most plasma supernatant isremoved and enough is left to resuspend the platelets. Platelets areessential for blood clotting however they also contain valuable growthfactors useful for cell culture, proliferation and survival. To releasethese factors, platelets should be lysed. There are several ways to lyseplatelets. One example is by repeated cycles of freezing at −80° C. forhours and thawing at 37° C. To remove platelets membranes, the lysate isspun at 4,000 rpm for 30 min at room temperature in a centrifuge and thesupernatant is transferred to another sterile tube. Serum, plasma,platelets lysate can be used right away or stored, and typically will beused right away to culture and store the other products.

The cord tissue is cleaned and cut into two portions, or it can be cutinto portions first. The first portion is for the purpose of collectingall the cells inside the umbilical cord, and the second one for thepurpose of obtaining a decellularized matrix. As one alternative, wecould also use the same piece for both processes, when we mince thetissue or slice it fine enough to liberate the cells with gentleagitation. However, typically we use different portions for the twoproducts.

The sterilizing agent can be selected from those known in the field,including hydrogen peroxide, isopropyl alcohol, ethanol or CholaPrep™from Becton Dickinson.

In order to make a cell mixture, the cord is typically minced, althoughthin slices could be made as well, and the cord gently agitated or flowpassed over the tissue to gently release cells, which are collected bycentrifugation, sedimentation or filtration. If the cord is very thinlysliced or spiral sliced, the sheets can be gently agitated to releasethe cells, and the remaining cell free sheets can then be separated fromthe cells and used to make decellularized matrix.

The cell mixture is combined with the autologous serum, platelets orlysate, although syngeneic or HLA and gender matched product could beused instead. It is also possible to pool these blood products for use.The combination of mixed cells and serum, platelets or lysate can eitherbe stored by freezing, or they can be cultured before storage, eitherwith or without the decellularized matrix, as desired.

To make decellularized matrix, the cord portion is minced or cut into adesired shape either before or after decellularization, or both. Thenthe tissue is decellularized, using enzymatic, chemical, osmotic ormechanical means, but preferably avoiding harsh chemicals or enzymesthat might be difficult to eliminate or might change the matrix in anyway. A large numbers of washes ensures that the decellularized matrix isfree of any reagents, and the matrix can be frozen, or freeze dried, orused right away. Further, it can be frozen with cord cell mixturesand/or serum, etc., or not, as desired. The resulting decellularizedtissue product can be further cut into desired shape that allows theflexibility for future 3D culturing and/or differentiation of stem cellsunder various circumstances for tissue engineering or regenerativetherapies.

FIG. 4A is a side view of how to mechanically slice cord tissue beforeor after decellularization. FIG. 4B is a cross-section of the umbilicalcord, illustrating an example of the cutting/paring path around thevessels. The umbilical cord is spirally sliced along an axis from thesurface and at a desired depth with a blade or laser. As the paringprogresses, the continuous paring path is illustrated in FIG. 4B, wherethe eventual cord matrix sheet has portions with vessel wall andportions without.

In a Class 100 or 10,000 environment, the cord is straightened orelongated by placing a long glass or metal rod in the vessels or thecord itself or both. Next, the rod is rotated over a cutting razor orlaser. Adjusting the depth of the blade or laser allows us to producematrices of different thicknesses as the cord rotates over the blade orlaser, or vice versa. Consequently, sheets of cut cord are prepared andpackaged in sterile packages. As an alternate method, the cord can beembedded on e.g., agar or frozen and then spiral sliced, these processeshelping to support the cord as it is cut.

Alternatively, as exemplified in FIG. 4A-I, before or afterdecellularization, the cord can be cut along several planes to producedifferent cord matrix shapes. FIGS. 4A and 4B depict how a cord tissuecan be sliced in sheets as the cord tissue rotates over a cutting bladeor knife. The cord can also be fixed then a knife or blade cuts the cordtissue while rotating around it. FIG. 4A is a lateral view of a wholeintact umbilical cord devoid of blood and without removing blood vesselsit is sliced with a knife, blade or laser from the surface to produce asheet of tissue of certain depth. FIG. 4B is a cross section ortransverse view of the umbilical cord showing the cutting path thatspirals from the surface inward although this spiral cutting can be madein reverse where the cord can be cored or cut from the inside towardsthe surface. In any case the sheet of cord retains blood vessel walls orportions of blood vessel walls. Cord tissue can also be frozen orembedded before being cut. FIG. 4C is an illustration of differentcutting angles along the umbilical cord. FIG. 4D-4I show examples ofdifferent cord tissue cutting angles or paths showed by dotted or dashedlines. Figure on far left shows examples of umbilical cord tissuecutting planes before or after decellularization. FIG. 4D is a randommince of tissue. FIG. 4E is a cross section. FIG. 4F is an oblique cut.FIG. 4G is a longitudinal cut. FIG. 4H is a curved cut. FIG. 4I is acylindrical or circular cut resulting in a cord piece with or withoutwhole vessels and with or without the original cord surface. Theseshapes range from a collection of small of tissue matrix particles (likea slurry) to large matrix-intact decellularized cord tissue pieces inthe shape of sheet, blocks, and the like.

The cell mixture can also be cultured together with the decellularizedtissue. The decellularized tissue serves as a 3D scaffold the same aswhat the cells experienced in vivo, thus more closely mimicking actualcell growth. Likewise, the oxygen concentration is adjusted to a rangebetween 0.1% and 7% at 37° C., like the in vivo condition. The culturemedium is supplemented with the serum, plasma or platelet lysateobtained from autologous or syngeneic cord blood or from an HLA- andsex-matched blood, therefore reducing the incompatibility issue and therisk of cross contamination.

It is to be noted that the isolated cells need not be culturedimmediately after collection, because the need for such culturing maynot have arisen yet. The isolated cell mixture or matrix can bepreserved for private or public use until such time that a matchingpatient needs treatment. Further, blood samples can be collected fromthe donor over time to provide a sufficient source ofserum/plasma/platelet lysates to supplement future cell culturing ortransplantations.

It is also to be noted that the cell mixture and decellularized tissuematrices need not be cultured immediately after collection, because theneed for such products may also not have arisen yet. The isolated cellmixture can be preserved for public use until such time that an HLA- andor sex matched patient needs treatment, or it can be preserved forprivate use by the individual that provided the cord tissue at birth.Further, HLA- and optionally sex matched blood samples can be collectedfrom the same HLA- and optionally sex matched donors to provide asufficient source of serum/plasma/platelet lysates to supplement futurecell culturing or transplantations. Alternatively, an individual canprovide his own autologous blood product for use in culturing the mixedcells and/or matrix, since when the need arises, collection of bloodwill typically become practical as most often the cells will not beneeded until adulthood.

Methods

Intact cord is collected and sterilized by flushing cord vessels withsterile antibiotic and antimycotic containing calcium- andmagnesium-free PBS. The cord surface is cleaned with regular alcoholswabs before placing in a sterile bag or container containing calcium-and magnesium-free PBS supplemented with antibiotics and antimycotics,sealed and safely placed in a 2° C. to 10° C. compartment of atemperature controlled damage resistant shipping box, shipped to thelaboratory, preferably a hybrid facility and received no more than 16hours post delivery. At the hybrid facility, private and public cordtissues are processed independently in designated areas in differentways. Cord blood is also collected and shipped to the same facility.

For cryopreserving whole cord tissue, a piece of cord tissue is cut; asterile plastic tube is inserted in the vessels to keep them distendedor straight; the tissue is placed in a sterile tube filled with lowglucose Dulbecco Modified Essential Medium (DMEM) or similar medium(e.g., a variety of stem cell media are commercially available)containing antibiotics and antimycotics, such as penicillin andstreptomycin respectively, with or without 5-20% final concentration ofserum, plasma or platelet lysate derived from cord blood of the same ordifferent newborn. GMP grade cryoprotectant is added to a finalconcentration of 1%-10% before quick or slow freezing to below −120° C.then quarantined in the gas phase of liquid nitrogen until communicabledisease diagnostics is clear. At this point, the tissue is transferredto long-term gas phase liquid nitrogen dewars designated for eitherprivate or public umbilical cord donors.

To manufacture an acellular matrix that can be used in the future forautologous or allogeneic purposes to support the regeneration of bone,cartilage, skin, fat, muscle, retinal, lens and nervous tissue, a pieceof cord tissue is straightened by inserting a thick glass stick in thecord vessels. Then the cord tissue is decellularized with 0.1% peraceticacid (PAA) for 2 hours with mechanical agitation and subsequent 15minutes wash with phosphate buffered saline (PBS) before treating thetissue with DNAse and RNAse in calcium magnesium-free PBS at 37° C. for1 hour. Cord tissue can also be decellularized with other chemicalreagents, such as hypotonic and hypertonic solutions, alcohols, SDS orother ionic or non-ionic detergents, trypsin, and the like. Mechanicaldissociation methods can also be used, such as convection flow,sonication, and the like. Decellularized matrix can be derived from aportion of cord tissue that was previously cut into desired shape with ablade or laser, or triturated, or homogenized followed bydecellularization with or without inserting a stick in the vessels.Therefore, acellular matrix sizes range from small particles to varioussizes and shapes of cord tissue, including a natural shape, sheets,blocks, and the like.

Cord tissue matrix is then washed with calcium magnesium-free PBS andpreserved by placing it low temperature resistant membranes orcontainers containing calcium magnesium-free PBS or medium supplementedor not with antibiotics and antimycotics, 1%-10% cryoprotectionsolution, plus 1-10% of autologous, syngeneic or HLA- and sex-matchedserum, plasma or platelet lysate. The matrix and serum combination isthen slowly and gradually or fast snap frozen to −20° C. or below.

In contrast, when the cells are to be retained, the tissue can be finelysliced, diced or homogenized without removing surface membrane orvessels and the cells gently liberated. For example, the tissue can becompletely sliced into thin strips (shaved slices), or only striatedpartway through, and gentle rocking or convection flow applied, ifneeded, to free the cells from the thin piece of tissue. Cells insolution are collected and separated from tissue matrix by filtration,sedimentation or density gradient centrifugation. Cells can also bedissociated by homogenizing whole cord tissue using a mortar and pestleor a commercially available device. This method preserves the cells forsubsequent use.

To isolate novel mesenchymal stem cells (along with progenitor anddifferentiated cells and other not yet characterized cells likenon-adherent or not yet surface adhering cells), cord tissue ismechanically dissociated or cut within 16 hours of delivery into thinfilaments (including vessel walls) using a sterile scalpel orhomogenizer and then liberated single and clumped cells still attachedto some cord tissue matrix are directly frozen slowly or quickly tobelow −120° C. in medium containing 1%-10% cryoprotectant preferablysupplemented with serum, plasma or platelet lysate derived from the samenewborn. Alternatively, isolated single cells and clumps of cells stillattached to some cord tissue matrix are placed in suspension to becultured for days or months in a prior art 3D chamber, preferably in0.5%-7% oxygen pressure, humidified CO₂ and N₂ gas environment and a lowglucose DMEM medium, preferably supplemented with either serum, plasmaor platelet lysate derived from the same newborn. The cells can also becultured for days or months on a cord tissue-derived biomatrix having acertain desired 3D shape. Blood components can also be isolated from thesame person or animal later in life, thus maintaining a source ofautologous blood components as long as possible.

Although there exists a controversy over the expression of “mesenchymalstem cells” markers, the isolated cord tissue-derived cells contains apool of cells with the following surface markers when grown in2-dimensional culture in the presence of fetal bovine serum: CD10⁺,CD29⁺⁺⁺, CD44⁺⁺⁺, CD73⁺⁺, CD54⁺, CD58⁺, CD105⁺, low CD106⁺, CD146⁺+,CD166⁺, low HLA-ABC⁺, STRO-1⁺. Gatta V. et. al., (2013) Gene expressionmodifications in Wharton's Jelly mesenchymal stem cells promoted byprolonged in vitro culturing. BMC Genomics 2013, 14:635; Arutyunyan I.et. al., (2016) Umbilical Cord as Prospective Source for MesenchymalStem Cell-Based Therapy. Stem Cells International Article ID 6901286.

The different plus signs define the degree of a marker's expressionwhereby one plus sign means little expression and more plus signs meanhigher expression. Another isolated pool of cord tissue-derived cells donot have the following surface markers when grown in 2-dimensionalculture in the presence of fetal bovine serum: CD3⁻, CD7⁻, CD19⁻, CD14⁻,CD28⁻, CD31⁻, CD33⁻, CD34⁻, CD38⁻, CD40⁻, CD45⁻, CD56⁻, CD62L⁻, CD62P⁻,CD80⁻, CD86⁻, CD90⁻, CD106⁻, CD117⁻, CD133⁻, CD135⁻, CD144⁻, CD271⁻,CD326⁻, HLA-DR⁻. Lin C. S., Ning H., Lin G., Lue T. F. (2012) Is CD34truly a negative marker for mesenchymal stromal cells? Cytotherapy14:1159-1163. Imran Ullah I. et. al., (2015) Human mesenchymal stemcells—current trends and future prospective. Bioscience Reports35/art:e00191. Still another pool of isolated cord tissue-derived cellshas the following markers: CD31, CD34, CD105, CD146, VE-Cadherin,VEGFR1, VEGFR2, Tie2, CXCR4, Von Willebrand, Aldehyde dehydrogenase butdoes not express CD1, CD115, CD45 and CD133. However, with our cellculture methods we expect these cells to have a different molecularmarker signature and/or strength of marker expression. These surfacemarkers can be used to further characterize and/or isolate cells ofvarious differentiation potentials when cultured in 3D and/or in thepresence of 0.5%-7% (or preferably 0.5%-5%) oxygen and/or in thepresence of serum, plasma or platelet lysate derived from an HLA- andoptionally sex-matched animal.

The cord blood, tissue cells and tissues biomatrix may be pooled frommore than one donors, as long as the donors are HLA- and optionallysex-matched. The process is shown in FIG. 7 that shows an example ofumbilical cord blood, cord tissue cells and cord tissue matrix productspooled from three HLA- and optionally sex-matching umbilical cords. InStep 701, three umbilical cord from three HLA-matching and optionallysex-matching donor animals are obtained. Each cord is then cut into twoportions, and the cord blood from each cord is processed. Anticoagulatedblood is first collected from each cord then processed into serum orplasma and blood stem cell bag products. From each cord blood processed,collect: one red blood cell (RBC) bag product; one plasma bag product;one plasma- & RBC-reduced cord blood bag product, and then pool eachHLA- and optionally sex-matched product type into one bag.

In Step 703, portions for deriving cells are pooled together, bymechanical dissociation of tissue to extract all cells from the firstcord portion. Example of pooled HLA- and/or sex-matched cordtissue-derived cells product is shown, and optionally being frozen insterile vials.

In step 705, portions for deriving matrices are pooled together, byextracting biomatrix from the second cord portion. Example of PooledHLA- and/or sex matched 3D decellularized cord tissue biomatrix productthat can be further cut into desired shape, and optionally frozen insterile container.

In some instances where a patient has not saved his or her umbilicalcord, or when a newborn is affected by a genetic disease, it is best totreat that individual with cell and matrix products derived from HLAmatching and preferably sex matching donors. The greater the HLA and sexmatch the greater the chance of engraftment and tolerance oftransplanted cells, matrices and tissues.

Before or after culture, HLA- and optionally sex-matched cells andmatrices cultured alone or in combination are collected and a sample ischaracterized by some or all of the following: sterility, size, cellsurface and intracellular markers, cell proliferation rate, cellself-renewal and differentiation potential, matrix strength and cellinfiltration, using microscopy, flow cytometry, cell and molecularbiology techniques, as well as engraftment and regeneration assays inanimal models. Concurrently, all cells are collected, washed andsuspended in fresh medium containing HLA and/or sex matched cord bloodserum, plasma or platelet lysate and 1% to 10% GMP grade cryoprotectantbefore slow and gradual or fast freezing to −120° C. or below, thenquarantined in the gas phase of liquid nitrogen until communicabledisease diagnostics is clear. Until then, the cells or tissues aretransferred to long-term gas phase liquid nitrogen dewars designated foreither private or public umbilical cord donors.

On use, the cells can be allowed to attach or are reinfused back in thedecellularized tissue biomatrix of a desired shape. This is done bycombining the two using varying concentration of cells anddecellularized tissue biomatrices and allowing the cells to attach anddiffuse into the matrix, or by injecting them thereinto, depending onmatrix shape and size. The cells can be used alone, but thedecellularized tissue matrix provides a useful 3D scaffold and aregulated environment for stem cell maintenance and growth, and is apreferred methodology. Umbilical cord derived cells and decellularizedtissue matrix can be used alone or in any combinations for animal orhuman therapeutic purposes. See FIG. 1, FIG. 2, Table 1, Table 2, Table3, Table 4, and Table 5.

Although various embodiments of the method and apparatus of the presentdisclosure have been illustrated in the accompanying Drawings (FIGS. 1through 7 and Tables 1 through 5) and described in the foregoingDetailed Description, it will be understood that the disclosure is notlimited to the embodiments disclosed, but is capable of numerousrearrangements, modifications and substitutions without departing fromthe spirit of the disclosure as set forth herein.

The following are each incorporated by reference herein in its entiretyfor all purposes:

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TABLE 1 Each row refers to the product(s) marked by “X” delivered beforeor after 3D culture for research or AUTOLOGOUS PRODUCTS FROM ONE DONORtransplantation in an Serum, Plasma or Umbilical Cord Umbilical CordWhole Umbilical HLA− and/or sex Platelet Lysate Blood Cells from TissueCells from Cord Tissue Matrix matches patient from newborn A newborn Anewborn A from newborn A 1 X DELIVERED BEFORE OR 2 X X AFTER 3D CULTUREIN 3 X X X OR WITHOUT HYPOXIA 4 X X X X FOR RESEARCH OR 5 X X XTRANSPLANTATION IN 6 X X AN HLA− AND/OR SEX 7 X X MATCHED PATIENT 8 X XX 9 X 10 X X 11 X X 12 X X X 13 X 14 X 15 X X

TABLE 2 Each row refers to the product(s) marked by “X” delivered beforeor Allogeneic Product after 3D culture for Pooled HLA− and/or researchor AUTOLOGOUS PRODUCTS FROM ONE DONOR Sex Matching Whole transplantationin an Serum, Plasma or Umbilical Cord Umbilical Cord Umbilical CordTissue HLA− and/or sex Platelet Lysate Blood Cells from Tissue Cellsfrom Matrix (taken from matched patient from newborn A newborn A newbornA donors) 1 X DELIVERED BEFORE OR 2 X X AFTER 3D CULTURE IN 3 X X X ORWITHOUT HYPOXIA 4 X X X X FOR RESEARCH OR 5 X X X TRANSPLANTATION IN 6 XX AN HLA− AND/OR SEX 7 X X MATCHED PATIENT 8 X X X 9 X 10 X X 11 X X 12X X X 13 X 14 X 15 X X

TABLE 3 Each row refers to the Allogeneic Product product(s) marked by“X” Pooled HLA− and/or delivered before or after 3D Sex matching sera/AUTOLOGOUS PRODUCTS FROM ONE DONOR culture for research or plasmas orplatelet Umbilical Cord Umbilical Cord Whole Umbilical transplantationin an HLA− lysates (taken from Blood Cells from Tissue Cells from CordTissue Matrix and/or sex matched patient donors) newborn A newborn Afrom newborn A 1 X DELIVERED BEFORE OR 2 X X AFTER 3D CULTURE IN 3 X X XOR WITHOUT HYPOXIA 4 X X X X FOR RESEARCH OR 5 X X X TRANSPLANTATION IN6 X X AN HLA− AND/OR SEX 7 X X MATCHED PATIENT 8 X X X 9 X 10 X X 11 X X12 X X X 13 X 14 X 15 X X

TABLE 4 Allogeneic Product Each row refers to the Allogeneic ProductPooled HLA− and/or product(s) marked by “X” Pooled HLA− and/orAutologous Products from Same Sex matching Whole delivered before orafter 3D Sex matching sera/ Donor Umbilical Cord Tissue culture forresearch or plasmas or platelet Umbilical Cord Umbilical Cord Matrix(taken from transplantation in an HLA− lysates (taken from Blood Cellsfrom Tissue Cells from same pool of sera and/or sex matched patientdonors) newborn A newborn A donors) 1 X DELIVERED BEFORE OR 2 X X AFTER3D CULTURE IN 3 X X X OR WITHOUT HYPOXIA 4 X X X X FOR RESEARCH OR 5 X XX TRANSPLANTATION IN 6 X X AN HLA− AND/OR SEX 7 X X MATCHED PATIENT 8 XX X 9 X 10 X X 11 X X 12 X X X 13 X 14 X 15 X X

TABLE 5 Each row refers to the Pooled Pooled Umbilical Pooled HLA−and/or product(s) marked by HLA− and/or Cord Blood Umbilical Cord TissueSex Matching Whole “X”, delivered before or Sex matching Cells from HLA−Cells HLA− and/or Sex Umbilical Cord Tissue after 3D culture forsera/plasmas and/or Sex matched matched patient Matrix (taken from sameresearch or transplantation or platelet patient (taken from (taken fromsame pool pool of sera and cord in an HLA− and/or lysates (taken samepool of sera of sera and cord blood blood and cord tissue sex matchedpatient from donors) donors) donors) cells donors) 1 X DELIVERED BEFOREOR 2 X X AFTER 3D CULTURE IN 3 X X X OR WITHOUT HYPOXIA 4 X X X X FORRESEARCH OR 5 X X X TRANSPLANTATION IN 6 X X AN HLA− AND/OR SEX 7 X XMATCHED PATIENT 8 X X X 9 X 10 X X 11 X X 12 X X X 13 X 14 X 15 X X

I claim:
 1. A biomatrix, said biomatrix obtained by steps consistingessentially of: a) obtaining a first intact portion of umbilical cordtissue; b) cleaning said first intact portion of umbilical cord tissuewith a sterilizing agent; c) decellularizing said first intact portionof umbilical cord tissue to produce a decellularized tissue; d) cuttingsaid decellularized tissue to a desired shape without removing surfacemembranes or blood vessels; e) incubating said decellularized tissuewith serum, plasma or platelet lysate in 0.1% to 5% oxygen environment;and f) optionally recombining said decellularized tissue from step ewith cells.
 2. The biomatrix of claim 1, wherein said serum, plasma orplatelet lysate in step e) is autologous or syngeneic or HLA-matched tosaid umbilical cord tissue having at least 3 matched MHC loci.
 3. Thebiomatrix of claim 1, wherein step f) is not optional and saiddecellularized tissue is combined with i) mononuclear cells from cordblood, or ii) a mixture of cells mechanically isolated from a secondintact portion of umbilical cord tissue, or both i) and ii), whereinsaid cord blood and said mixture of cells are autologous, syngeneic orHLA-matched to said umbilical cord tissue with the same at least 3matched MHC loci.
 4. The biomatrix of claim 3, wherein one or more of i)said mononuclear cells, ii) said cell mixture iii) said serum, plasma orplatelet lysate, or iv) said decellularized tissue is a pooledHLA-matched product, each pooled portion of said HLA-matched producthaving the same at least 3 matched MHC loci.